{"gene":"EHD3","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2002,"finding":"EHD3 localizes to endocytic vesicles and microtubule-dependent membrane tubules, colocalizing with transferrin-containing recycling vesicles; the N-terminal domain is responsible for tubular localization. EHD1 and EHD3 interact via two-hybrid analysis and co-immunoprecipitation from cellular extracts, and coexpression results in colocalization in microtubule-dependent tubules.","method":"GFP-fusion localization, N-terminal domain swapping/deletion mutagenesis, yeast two-hybrid, co-immunoprecipitation","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus two-hybrid plus domain mutagenesis in a single study from one lab","pmids":["12121420"],"is_preprint":false},{"year":2005,"finding":"EHD3 binds the Rab11-effector Rab11-FIP2 via EH domain–NPF motif interactions; this association is affected by nucleotide-binding status. Knockdown of EHD3 prevents delivery of internalized transferrin and early endosomal proteins to the endocytic recycling compartment (ERC), demonstrating a role for EHD3 in early endosome-to-ERC transport distinct from EHD1's role in ERC exit.","method":"Co-immunoprecipitation, siRNA knockdown, transferrin recycling assay, subcellular localization imaging","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assay with nucleotide-binding mutants plus functional siRNA knockdown with specific trafficking readout, single lab but multiple orthogonal methods","pmids":["16251358"],"is_preprint":false},{"year":2007,"finding":"Rab8a and Myosin Vb colocalize to a tubular network containing EHD1 and EHD3 (distinct from Rab11a-containing compartments), as demonstrated by live-cell FRET imaging and co-localization studies.","method":"Yeast two-hybrid, FRET, live-cell imaging, co-localization","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET-confirmed interaction and live imaging, but EHD3 role is indirect (co-localization marker); single lab","pmids":["17507647"],"is_preprint":false},{"year":2009,"finding":"siRNA knockdown of EHD3 (or its interaction partner rabenosyn-5) redistributes sorting nexin 1 to enlarged early endosomes, disrupts Shiga toxin B subunit transport to the Golgi, fragments Golgi morphology, reduces AP-1 gamma-adaptin recruitment to the Golgi, misroutes mannose 6-phosphate receptor to peripheral endosomes, and traps cathepsin D at the Golgi — establishing EHD3 as a regulator of early-endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking.","method":"siRNA knockdown, immunofluorescence, Shiga toxin transport assay, VSV-G secretion assay, mannose 6-phosphate receptor trafficking assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal trafficking assays after clean siRNA knockdown, single lab with strong mechanistic depth","pmids":["19139087"],"is_preprint":false},{"year":2011,"finding":"Mice doubly deficient for EHD3 and EHD4 develop thrombotic microangiopathy-like glomerular lesions with altered VEGFR2 expression and localization in glomerular endothelium and increased apoptosis, suggesting EHD3/EHD4-mediated endocytic recycling of VEGFR2 is essential for glomerular endothelial homeostasis.","method":"Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models, histopathology, immunofluorescence, proteinuria measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO model with defined phenotype and receptor localization data, but mechanistic link to VEGFR2 trafficking is partially inferential; single lab","pmids":["21408024"],"is_preprint":false},{"year":2013,"finding":"siRNA knockdown or doxycycline-inducible restoration of EHD3 in glioma cell lines shows EHD3 decreases cell growth and invasiveness, induces cell cycle arrest and apoptosis; promoter hypermethylation silences EHD3 expression and is reversible by 5-Azacytidine; xenograft experiments confirm in vivo tumor suppressive activity.","method":"siRNA knockdown, doxycycline-inducible overexpression, bisulfite sequencing, 5-Azacytidine demethylation, xenograft mouse model, cell cycle/apoptosis assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell-based and in vivo methods from a single lab establishing a functional role, but molecular mechanism of growth suppression not fully dissected","pmids":["24306026"],"is_preprint":false},{"year":2013,"finding":"siRNA depletion of EHD3 in HeLa cells delays short-loop β3-integrin recycling from early endosomes back to the cell surface and impairs αvβ3-integrin-mediated cell adhesion. TIRF-based colocalization shows β3-integrin transits EHD3-positive endosomes near the cell surface, consistent with a rapid-recycling role.","method":"siRNA knockdown, live-cell TIRF microscopy, integrin recycling assay, cell adhesion assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization and functional recycling assay with TIRF, single lab","pmids":["23781025"],"is_preprint":false},{"year":2014,"finding":"EHD3-deficient mouse hearts display bradycardia, conduction block, and blunted adrenergic response. EHD3-deficient myocytes have reduced membrane expression/localization of Na/Ca exchanger (NCX1) and L-type Ca channel Cav1.2, reduced corresponding membrane currents, increased sarcoplasmic reticulum Ca2+ and spark frequency, and reduced ankyrin-B expression/localization. Ankyrin-B co-immunoprecipitates with EHD3 and NCX1, placing EHD3 in an endosome-based trafficking pathway for these cardiac membrane proteins.","method":"Cardiac-specific EHD3 KO mouse, electrophysiology, patch-clamp, immunofluorescence/confocal microscopy, co-immunoprecipitation, Ca2+ spark imaging","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo cardiac-specific KO combined with electrophysiology, Ca2+ imaging, Co-IP, and membrane protein localization in a single rigorous study","pmids":["24759929"],"is_preprint":false},{"year":2012,"finding":"EHD3 protein levels are consistently elevated in four different heart failure models (ischemic rat, pressure-overload mouse, pacing-induced canine, and failing human myocardium); NCX1 levels parallel EHD3 upregulation. EHD3 upregulation in heart failure is downstream of reactive oxygen species and angiotensin II signaling.","method":"Western blot across multiple HF models, ROS and angiotensin II pharmacological manipulation","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple independent HF models replicate the finding but mechanistic link to Ang II/ROS is pharmacological without genetic dissection","pmids":["22406195"],"is_preprint":false},{"year":2015,"finding":"EHD3 undergoes SUMO modification at lysines K315 and K511, both in vitro and in cells. SUMOylation is required for EHD3 localization to tubular ERC structures; non-SUMOylated EHD3 acts as a dominant negative for tubulation and delays transferrin recycling from the ERC to the cell surface. SUMOylation does not affect EHD3 dimerization.","method":"In vitro SUMOylation assay, site-directed mutagenesis of SUMO acceptor lysines, transferrin recycling assay, immunofluorescence","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of SUMOylation plus mutagenesis plus functional recycling assay in cells, single lab with multiple orthogonal methods","pmids":["26226295"],"is_preprint":false},{"year":2015,"finding":"EHD1 and EHD3, together with the Rab11–Rab8 cascade, localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is required to form ciliary vesicles from distal appendage vesicles (DAVs) at the mother centriole, a step necessary for basal body transformation, transition zone protein recruitment, and IFT20 recruitment before ciliary growth. SNAP29 (a SNARE and EHD1-binding protein) is also required for this DAV-to-ciliary-vesicle fusion step.","method":"siRNA knockdown, super-resolution and electron microscopy, live imaging, co-immunoprecipitation, rescue experiments","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA knockdown plus EM ultrastructure plus live imaging plus Co-IP, replicated across multiple experimental approaches in one rigorous study","pmids":["25686250"],"is_preprint":false},{"year":2016,"finding":"EHD3 stabilizes tubular recycling endosomes (TRE) rather than initiating their biogenesis; in a synchronized TRE regeneration assay (phospholipase D inhibitor washout), EHD3 depletion did not prevent TRE formation but shortened their persistence. The residues Asn-519/Glu-520 in EHD3's EH domain (vs. Ala-519/Asp-520 in EHD1) define the differential selectivity of these paralogs for NPF-containing binding partners, explaining distinct roles: EHD1 in vesiculation vs. EHD3 in tubule stabilization.","method":"siRNA knockdown, phospholipase D inhibitor washout assay, site-directed EH domain mutagenesis, co-localization imaging","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — synchronized TRE biogenesis system plus EH domain mutagenesis identifying specific residues, multiple orthogonal methods in single rigorous study","pmids":["27189942"],"is_preprint":false},{"year":2016,"finding":"EHD3 binds phosphatidic acid (PA) through its helical domain, as shown by in vitro liposome co-sedimentation. PA–EHD3 interaction induces liposomal tubulation in vitro. Inhibiting PA synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor reduces EHD3-containing tubules and impairs early endosomal trafficking, establishing that PA cooperates with EHD3 to drive membrane tubulation.","method":"In vitro liposome co-sedimentation assay, pharmacological PA synthesis inhibition, immunofluorescence tubule counting","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of PA-induced tubulation plus pharmacological loss-of-function in cells, single lab with orthogonal methods","pmids":["26896729"],"is_preprint":false},{"year":2016,"finding":"EHD3 accelerates EGFR degradation upon EGF stimulation by increasing EGFR ubiquitination and diverting EGFR trafficking from the recycling route to the degradative pathway. EHD3 reduces endosome-based MAPK and AKT signaling downstream of EGFR without affecting total pathway activity, demonstrating spatial regulation of EGFR signaling.","method":"Doxycycline-inducible EHD3 expression, EGFR ubiquitination assay, endosomal trafficking/recycling assays, immunofluorescence, western blot for pathway activation","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple trafficking and signaling assays in a single lab, but no in vitro reconstitution of ubiquitination mechanism","pmids":["27811356"],"is_preprint":false},{"year":2021,"finding":"NR5A1 transcriptionally activates EHD3 by binding the conserved 'AGGTCA' sequence in the EHD3 promoter. EHD3 overexpression increases testosterone concentration; EHD3 knockdown decreases testosterone synthesis by reducing endocytosis in Leydig cells. Leydig-cell-specific NR5A1 knockout mice show reduced EHD3, clathrin, and serum testosterone levels.","method":"ChIP, dual luciferase reporter assay, siRNA knockdown, exosome tracing/endocytosis assay, conditional NR5A1 KO mouse (CRISPR/Cas9), ELISA","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-validated transcription factor binding plus in vivo conditional KO, single lab","pmids":["33964295"],"is_preprint":false}],"current_model":"EHD3 is an EH domain-containing ATPase that localizes to early endosomes and tubular recycling endosomes (TRE), where it functions downstream of a Rab11–Rab8 cascade to mediate early endosome-to-ERC transport, endosome-to-Golgi trafficking, and ciliogenesis initiation via membrane tubulation; it binds phosphatidic acid through its helical domain to drive tubule formation, is SUMOylated on K315/K511 to maintain TRE localization and stability, heterodimerizes with EHD1 through specific EH domain residues (N519/E520) that distinguish its tubule-stabilizing role from EHD1's vesiculation activity, interacts with Rab11-FIP2 and ankyrin-B to traffic cargo including NCX1, Cav1.2, and αvβ3-integrin, and diverts EGFR toward degradative trafficking to attenuate endosomal signaling."},"narrative":{"mechanistic_narrative":"EHD3 is an EH domain-containing membrane-remodeling protein that drives the formation and stabilization of tubular recycling endosomes and governs cargo transit through the early-endosomal system [PMID:12121420, PMID:27189942]. It binds phosphatidic acid through its helical domain, and this lipid interaction is sufficient to deform liposomes into tubules in vitro and is required for EHD3-positive tubules and early endosomal trafficking in cells [PMID:26896729]. Functionally, EHD3 mediates early endosome-to-endocytic recycling compartment (ERC) transport via an EH domain–NPF interaction with the Rab11 effector Rab11-FIP2, a role distinct from EHD1's function in ERC exit [PMID:16251358]; it also directs early endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking [PMID:19139087]. EHD3 heterodimerizes with EHD1 [PMID:12121420], and the EH domain residues Asn-519/Glu-520 specify its preference for NPF partners and explain its tubule-stabilizing role versus EHD1-driven vesiculation [PMID:27189942]; SUMOylation at K315 and K511 maintains its localization to tubular ERC structures and recycling activity [PMID:26226295]. Through these trafficking activities EHD3 controls surface delivery and recycling of specific cargo, including β3/αvβ3-integrin [PMID:23781025] and the cardiac membrane proteins NCX1 and Cav1.2 in an ankyrin-B–associated complex [PMID:24759929], and it diverts activated EGFR toward degradation to spatially attenuate endosomal MAPK/AKT signaling [PMID:27811356]. EHD3, together with EHD1 and the Rab11–Rab8 cascade, also generates ciliary vesicles from distal appendage vesicles at the mother centriole, a tubulation step required for ciliogenesis initiation [PMID:25686250].","teleology":[{"year":2002,"claim":"Established EHD3 as a membrane-tubulating protein of the recycling system and identified its physical partnership with EHD1, defining a molecular foundation for its trafficking role.","evidence":"GFP-fusion localization with domain swapping, yeast two-hybrid and Co-IP in cells","pmids":["12121420"],"confidence":"Medium","gaps":["Does not define the cargo handled by EHD3 tubules","Functional consequence of the EHD1–EHD3 interaction unresolved"]},{"year":2005,"claim":"Showed EHD3 binds the Rab11 effector Rab11-FIP2 via EH–NPF contacts and is required for early endosome-to-ERC delivery, distinguishing its trafficking step from EHD1.","evidence":"Co-IP with nucleotide-binding mutants, siRNA knockdown, transferrin recycling assay","pmids":["16251358"],"confidence":"High","gaps":["Structural basis of EH–NPF selectivity not resolved","How nucleotide state controls FIP2 binding mechanistically unclear"]},{"year":2007,"claim":"Placed EHD3 in a Rab8a/Myosin Vb tubular network distinct from Rab11a compartments, refining the membrane subdomain it occupies.","evidence":"FRET and live-cell imaging with co-localization","pmids":["17507647"],"confidence":"Medium","gaps":["EHD3 role is inferred from co-localization, not direct interaction","Functional contribution to this network untested"]},{"year":2009,"claim":"Extended EHD3 function beyond recycling to early endosome-to-Golgi transport, linking it to Golgi morphology and lysosomal enzyme delivery.","evidence":"siRNA knockdown with Shiga toxin, M6PR, and cathepsin D trafficking assays","pmids":["19139087"],"confidence":"High","gaps":["Direct partner mediating Golgi-directed step beyond rabenosyn-5 not fully defined","Whether tubulation per se drives this route untested"]},{"year":2012,"claim":"Connected EHD3 expression to cardiac stress, showing it is upregulated across heart failure models downstream of ROS and angiotensin II in parallel with NCX1.","evidence":"Western blot across multiple HF models with pharmacological ROS/Ang II manipulation","pmids":["22406195"],"confidence":"Medium","gaps":["Ang II/ROS link is pharmacological without genetic dissection","Transcriptional mechanism of upregulation unknown"]},{"year":2011,"claim":"Demonstrated in vivo physiological importance of EHD3 (with EHD4) in glomerular endothelial homeostasis via VEGFR2 recycling.","evidence":"Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models with histopathology and receptor localization","pmids":["21408024"],"confidence":"Medium","gaps":["Direct EHD3–VEGFR2 trafficking link is partly inferential","EHD3-specific vs EHD4-redundant contributions not separated"]},{"year":2013,"claim":"Identified EHD3 as a regulator of rapid β3-integrin recycling and αvβ3-mediated adhesion, defining a specific cargo of its short-loop recycling activity.","evidence":"siRNA knockdown, TIRF colocalization, integrin recycling and adhesion assays","pmids":["23781025"],"confidence":"Medium","gaps":["Adaptor coupling EHD3 to integrin not identified","Single lab, single cell type"]},{"year":2013,"claim":"Revealed a tumor-suppressive function for EHD3 silenced by promoter hypermethylation in glioma, broadening its role to growth and survival control.","evidence":"siRNA, inducible re-expression, bisulfite sequencing, 5-Aza demethylation, xenografts","pmids":["24306026"],"confidence":"Medium","gaps":["Molecular mechanism linking trafficking to growth suppression not dissected","Relationship to EGFR/signaling control unexplored here"]},{"year":2014,"claim":"Established EHD3 as essential for cardiac excitability through ankyrin-B–associated trafficking of NCX1 and Cav1.2 to the membrane.","evidence":"Cardiac-specific KO mouse, electrophysiology, Ca2+ imaging, Co-IP, localization","pmids":["24759929"],"confidence":"High","gaps":["Order of EHD3/ankyrin-B/cargo assembly unresolved","Whether tubulation activity is required for cardiac cargo delivery untested"]},{"year":2015,"claim":"Showed SUMOylation at K315/K511 is a post-translational switch controlling EHD3 ERC tubule localization and recycling capacity.","evidence":"In vitro SUMOylation, acceptor-lysine mutagenesis, transferrin recycling assays","pmids":["26226295"],"confidence":"High","gaps":["SUMO E3 ligase and signal triggering SUMOylation unknown","How SUMO controls tubule targeting mechanistically unclear"]},{"year":2015,"claim":"Defined EHD3's role in ciliogenesis initiation, generating ciliary vesicles from distal appendage vesicles via EHD-dependent tubulation within the Rab11–Rab8 cascade.","evidence":"siRNA, super-resolution and electron microscopy, live imaging, Co-IP, rescue","pmids":["25686250"],"confidence":"High","gaps":["EHD3-specific vs EHD1-redundant contribution to DAV fusion not separated","Direct membrane-fusion machinery coupling not fully mapped"]},{"year":2016,"claim":"Mechanistically distinguished EHD3 from EHD1 by showing it stabilizes pre-formed tubular recycling endosomes, with EH residues N519/E520 specifying NPF-partner selectivity.","evidence":"Synchronized TRE regeneration (PLD inhibitor washout), EH domain mutagenesis","pmids":["27189942"],"confidence":"High","gaps":["Structural model of N519/E520-dependent partner discrimination absent","TRE-stabilizing partners not enumerated"]},{"year":2016,"claim":"Provided the biochemical basis for EHD3 tubulation by showing its helical domain binds phosphatidic acid to deform membranes.","evidence":"Liposome co-sedimentation, PA synthesis inhibition, tubule quantification","pmids":["26896729"],"confidence":"High","gaps":["High-resolution structure of the PA-bound tubulating state lacking","Whether ATPase cycle couples to PA-driven tubulation untested"]},{"year":2016,"claim":"Showed EHD3 spatially regulates EGFR signaling by promoting receptor ubiquitination and degradative routing, attenuating endosomal MAPK/AKT output.","evidence":"Inducible expression, EGFR ubiquitination and trafficking assays, pathway westerns","pmids":["27811356"],"confidence":"Medium","gaps":["No in vitro reconstitution of the ubiquitination step","Ubiquitin ligase recruited by EHD3 not identified"]},{"year":2021,"claim":"Identified transcriptional control of EHD3 by NR5A1 and a physiological output in Leydig-cell testosterone synthesis via endocytosis.","evidence":"ChIP, luciferase reporter, siRNA, endocytosis assay, conditional NR5A1 KO mouse","pmids":["33964295"],"confidence":"Medium","gaps":["Specific endocytic cargo driving steroidogenesis not defined","Direct vs indirect contribution of EHD3 to testosterone synthesis unresolved"]},{"year":null,"claim":"How EHD3's ATPase cycle, PA binding, SUMOylation, and EH–NPF partner selection are integrated into a single tubule-stabilization mechanism, and how this distinguishes the diverse cargo-specific roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural/biochemical model of the tubulation cycle","Determinants of cargo selectivity across cell types unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,11,12]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0,1,3,6]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,10]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[3]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[10]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,7]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,3,6,7]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,3]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13]}],"complexes":[],"partners":["EHD1","RAB11FIP2","ANK2","NCX1","RAB8A","MYO5B","SNAP29"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NZN3","full_name":"EH domain-containing protein 3","aliases":["PAST homolog 3"],"length_aa":535,"mass_kda":60.9,"function":"ATP- and membrane-binding protein that controls membrane reorganization/tubulation upon ATP hydrolysis (PubMed:25686250). In vitro causes tubulation of endocytic membranes (PubMed:24019528). Binding to phosphatidic acid induces its membrane tubulation activity (By similarity). Plays a role in endocytic transport. Involved in early endosome to recycling endosome compartment (ERC), retrograde early endosome to Golgi, and endosome to plasma membrane (rapid recycling) protein transport. Involved in the regulation of Golgi maintenance and morphology (PubMed:16251358, PubMed:17233914, PubMed:19139087, PubMed:23781025). Involved in the recycling of internalized D1 dopamine receptor (PubMed:21791287). Plays a role in cardiac protein trafficking probably implicating ANK2 (PubMed:20489164). Involved in the ventricular membrane targeting of SLC8A1 and CACNA1C and probably the atrial membrane localization of CACNA1GG and CACNA1H implicated in the regulation of atrial myocyte excitability and cardiac conduction (By similarity). In conjunction with EHD4 may be involved in endocytic trafficking of KDR/VEGFR2 implicated in control of glomerular function (By similarity). Involved in the rapid recycling of integrin beta-3 implicated in cell adhesion maintenance (PubMed:23781025). Involved in the unidirectional retrograde dendritic transport of endocytosed BACE1 and in efficient sorting of BACE1 to axons implicating a function in neuronal APP processing (By similarity). Plays a role in the formation of the ciliary vesicle, an early step in cilium biogenesis; possibly sharing redundant functions with EHD1 (PubMed:25686250)","subcellular_location":"Recycling endosome membrane; Cell membrane; Cell projection, cilium membrane","url":"https://www.uniprot.org/uniprotkb/Q9NZN3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EHD3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EHD3","total_profiled":1310},"omim":[{"mim_id":"619563","title":"MICAL-LIKE PROTEIN 1; MICALL1","url":"https://www.omim.org/entry/619563"},{"mim_id":"612723","title":"PLECKSTRIN HOMOLOGY DOMAIN-CONTAINING PROTEIN, FAMILY H, MEMBER 2; PLEKHH2","url":"https://www.omim.org/entry/612723"},{"mim_id":"606540","title":"MYOSIN VB; MYO5B","url":"https://www.omim.org/entry/606540"},{"mim_id":"605892","title":"EH DOMAIN-CONTAINING 4; EHD4","url":"https://www.omim.org/entry/605892"},{"mim_id":"605891","title":"EH DOMAIN-CONTAINING 3; EHD3","url":"https://www.omim.org/entry/605891"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":41.0},{"tissue":"esophagus","ntpm":40.9}],"url":"https://www.proteinatlas.org/search/EHD3"},"hgnc":{"alias_symbol":[],"prev_symbol":["PAST3"]},"alphafold":{"accession":"Q9NZN3","domains":[{"cath_id":"1.10.268.20","chopping":"22-51_292-404","consensus_level":"high","plddt":93.0078,"start":22,"end":404},{"cath_id":"3.40.50.300","chopping":"61-285","consensus_level":"high","plddt":91.3733,"start":61,"end":285},{"cath_id":"1.10.238.10","chopping":"430-526","consensus_level":"high","plddt":90.8633,"start":430,"end":526}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NZN3-F1-predicted_aligned_error_v6.png","plddt_mean":88.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EHD3","jax_strain_url":"https://www.jax.org/strain/search?query=EHD3"},"sequence":{"accession":"Q9NZN3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NZN3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NZN3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NZN3"}},"corpus_meta":[{"pmid":"25686250","id":"PMC_25686250","title":"Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.","date":"2015","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/25686250","citation_count":211,"is_preprint":false},{"pmid":"16251358","id":"PMC_16251358","title":"Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16251358","citation_count":160,"is_preprint":false},{"pmid":"17507647","id":"PMC_17507647","title":"Myosin Vb interacts with Rab8a on a tubular network containing EHD1 and EHD3.","date":"2007","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/17507647","citation_count":141,"is_preprint":false},{"pmid":"21284756","id":"PMC_21284756","title":"Ehd3, encoding a plant homeodomain finger-containing protein, is a critical promoter of rice flowering.","date":"2011","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21284756","citation_count":138,"is_preprint":false},{"pmid":"10673336","id":"PMC_10673336","title":"EHD2, EHD3, and EHD4 encode novel members of a highly conserved family of EH domain-containing proteins.","date":"2000","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10673336","citation_count":80,"is_preprint":false},{"pmid":"19139087","id":"PMC_19139087","title":"EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology.","date":"2009","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/19139087","citation_count":79,"is_preprint":false},{"pmid":"12121420","id":"PMC_12121420","title":"EHD3: a protein that resides in recycling tubular and vesicular membrane structures and interacts with EHD1.","date":"2002","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/12121420","citation_count":74,"is_preprint":false},{"pmid":"17251388","id":"PMC_17251388","title":"Expression and subcellular distribution of novel glomerulus-associated proteins dendrin, ehd3, sh2d4a, plekhh2, and 2310066E14Rik.","date":"2007","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/17251388","citation_count":66,"is_preprint":false},{"pmid":"21408024","id":"PMC_21408024","title":"Renal thrombotic microangiopathy in mice with combined deletion of endocytic recycling regulators EHD3 and EHD4.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21408024","citation_count":41,"is_preprint":false},{"pmid":"24759929","id":"PMC_24759929","title":"EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology.","date":"2014","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/24759929","citation_count":31,"is_preprint":false},{"pmid":"22406195","id":"PMC_22406195","title":"Differential regulation of EHD3 in human and mammalian heart failure.","date":"2012","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/22406195","citation_count":31,"is_preprint":false},{"pmid":"27189942","id":"PMC_27189942","title":"EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27189942","citation_count":21,"is_preprint":false},{"pmid":"24306026","id":"PMC_24306026","title":"Ehd3, a regulator of vesicular trafficking, is silenced in gliomas and functions as a tumor suppressor by controlling cell cycle arrest and apoptosis.","date":"2013","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/24306026","citation_count":16,"is_preprint":false},{"pmid":"33964295","id":"PMC_33964295","title":"EHD3 positively regulated by NR5A1 participates in testosterone synthesis via endocytosis.","date":"2021","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33964295","citation_count":10,"is_preprint":false},{"pmid":"24607927","id":"PMC_24607927","title":"The gender-specific association of EHD3 polymorphisms with major depressive disorder.","date":"2014","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/24607927","citation_count":9,"is_preprint":false},{"pmid":"23781025","id":"PMC_23781025","title":"Αvβ3-integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid-recycling pathway.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23781025","citation_count":7,"is_preprint":false},{"pmid":"26226295","id":"PMC_26226295","title":"SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26226295","citation_count":6,"is_preprint":false},{"pmid":"26896729","id":"PMC_26896729","title":"Phosphatidic acid induces EHD3-containing membrane tubulation and is required for receptor recycling.","date":"2016","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/26896729","citation_count":5,"is_preprint":false},{"pmid":"27811356","id":"PMC_27811356","title":"Spatio-temporal regulation of EGFR signaling by the Eps15 homology domain-containing protein 3 (EHD3).","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27811356","citation_count":5,"is_preprint":false},{"pmid":"24997812","id":"PMC_24997812","title":"Association between EHD3 gene and the cognitive function of patients with major depressive disorder.","date":"2014","source":"Zhongguo yi xue ke xue yuan xue bao. 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EHD1 and EHD3 interact via two-hybrid analysis and co-immunoprecipitation from cellular extracts, and coexpression results in colocalization in microtubule-dependent tubules.\",\n      \"method\": \"GFP-fusion localization, N-terminal domain swapping/deletion mutagenesis, yeast two-hybrid, co-immunoprecipitation\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus two-hybrid plus domain mutagenesis in a single study from one lab\",\n      \"pmids\": [\"12121420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EHD3 binds the Rab11-effector Rab11-FIP2 via EH domain–NPF motif interactions; this association is affected by nucleotide-binding status. Knockdown of EHD3 prevents delivery of internalized transferrin and early endosomal proteins to the endocytic recycling compartment (ERC), demonstrating a role for EHD3 in early endosome-to-ERC transport distinct from EHD1's role in ERC exit.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, transferrin recycling assay, subcellular localization imaging\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assay with nucleotide-binding mutants plus functional siRNA knockdown with specific trafficking readout, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16251358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Rab8a and Myosin Vb colocalize to a tubular network containing EHD1 and EHD3 (distinct from Rab11a-containing compartments), as demonstrated by live-cell FRET imaging and co-localization studies.\",\n      \"method\": \"Yeast two-hybrid, FRET, live-cell imaging, co-localization\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET-confirmed interaction and live imaging, but EHD3 role is indirect (co-localization marker); single lab\",\n      \"pmids\": [\"17507647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"siRNA knockdown of EHD3 (or its interaction partner rabenosyn-5) redistributes sorting nexin 1 to enlarged early endosomes, disrupts Shiga toxin B subunit transport to the Golgi, fragments Golgi morphology, reduces AP-1 gamma-adaptin recruitment to the Golgi, misroutes mannose 6-phosphate receptor to peripheral endosomes, and traps cathepsin D at the Golgi — establishing EHD3 as a regulator of early-endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking.\",\n      \"method\": \"siRNA knockdown, immunofluorescence, Shiga toxin transport assay, VSV-G secretion assay, mannose 6-phosphate receptor trafficking assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal trafficking assays after clean siRNA knockdown, single lab with strong mechanistic depth\",\n      \"pmids\": [\"19139087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Mice doubly deficient for EHD3 and EHD4 develop thrombotic microangiopathy-like glomerular lesions with altered VEGFR2 expression and localization in glomerular endothelium and increased apoptosis, suggesting EHD3/EHD4-mediated endocytic recycling of VEGFR2 is essential for glomerular endothelial homeostasis.\",\n      \"method\": \"Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models, histopathology, immunofluorescence, proteinuria measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO model with defined phenotype and receptor localization data, but mechanistic link to VEGFR2 trafficking is partially inferential; single lab\",\n      \"pmids\": [\"21408024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"siRNA knockdown or doxycycline-inducible restoration of EHD3 in glioma cell lines shows EHD3 decreases cell growth and invasiveness, induces cell cycle arrest and apoptosis; promoter hypermethylation silences EHD3 expression and is reversible by 5-Azacytidine; xenograft experiments confirm in vivo tumor suppressive activity.\",\n      \"method\": \"siRNA knockdown, doxycycline-inducible overexpression, bisulfite sequencing, 5-Azacytidine demethylation, xenograft mouse model, cell cycle/apoptosis assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell-based and in vivo methods from a single lab establishing a functional role, but molecular mechanism of growth suppression not fully dissected\",\n      \"pmids\": [\"24306026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"siRNA depletion of EHD3 in HeLa cells delays short-loop β3-integrin recycling from early endosomes back to the cell surface and impairs αvβ3-integrin-mediated cell adhesion. TIRF-based colocalization shows β3-integrin transits EHD3-positive endosomes near the cell surface, consistent with a rapid-recycling role.\",\n      \"method\": \"siRNA knockdown, live-cell TIRF microscopy, integrin recycling assay, cell adhesion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization and functional recycling assay with TIRF, single lab\",\n      \"pmids\": [\"23781025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EHD3-deficient mouse hearts display bradycardia, conduction block, and blunted adrenergic response. EHD3-deficient myocytes have reduced membrane expression/localization of Na/Ca exchanger (NCX1) and L-type Ca channel Cav1.2, reduced corresponding membrane currents, increased sarcoplasmic reticulum Ca2+ and spark frequency, and reduced ankyrin-B expression/localization. Ankyrin-B co-immunoprecipitates with EHD3 and NCX1, placing EHD3 in an endosome-based trafficking pathway for these cardiac membrane proteins.\",\n      \"method\": \"Cardiac-specific EHD3 KO mouse, electrophysiology, patch-clamp, immunofluorescence/confocal microscopy, co-immunoprecipitation, Ca2+ spark imaging\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo cardiac-specific KO combined with electrophysiology, Ca2+ imaging, Co-IP, and membrane protein localization in a single rigorous study\",\n      \"pmids\": [\"24759929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EHD3 protein levels are consistently elevated in four different heart failure models (ischemic rat, pressure-overload mouse, pacing-induced canine, and failing human myocardium); NCX1 levels parallel EHD3 upregulation. EHD3 upregulation in heart failure is downstream of reactive oxygen species and angiotensin II signaling.\",\n      \"method\": \"Western blot across multiple HF models, ROS and angiotensin II pharmacological manipulation\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple independent HF models replicate the finding but mechanistic link to Ang II/ROS is pharmacological without genetic dissection\",\n      \"pmids\": [\"22406195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EHD3 undergoes SUMO modification at lysines K315 and K511, both in vitro and in cells. SUMOylation is required for EHD3 localization to tubular ERC structures; non-SUMOylated EHD3 acts as a dominant negative for tubulation and delays transferrin recycling from the ERC to the cell surface. SUMOylation does not affect EHD3 dimerization.\",\n      \"method\": \"In vitro SUMOylation assay, site-directed mutagenesis of SUMO acceptor lysines, transferrin recycling assay, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of SUMOylation plus mutagenesis plus functional recycling assay in cells, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26226295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EHD1 and EHD3, together with the Rab11–Rab8 cascade, localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is required to form ciliary vesicles from distal appendage vesicles (DAVs) at the mother centriole, a step necessary for basal body transformation, transition zone protein recruitment, and IFT20 recruitment before ciliary growth. SNAP29 (a SNARE and EHD1-binding protein) is also required for this DAV-to-ciliary-vesicle fusion step.\",\n      \"method\": \"siRNA knockdown, super-resolution and electron microscopy, live imaging, co-immunoprecipitation, rescue experiments\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA knockdown plus EM ultrastructure plus live imaging plus Co-IP, replicated across multiple experimental approaches in one rigorous study\",\n      \"pmids\": [\"25686250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EHD3 stabilizes tubular recycling endosomes (TRE) rather than initiating their biogenesis; in a synchronized TRE regeneration assay (phospholipase D inhibitor washout), EHD3 depletion did not prevent TRE formation but shortened their persistence. The residues Asn-519/Glu-520 in EHD3's EH domain (vs. Ala-519/Asp-520 in EHD1) define the differential selectivity of these paralogs for NPF-containing binding partners, explaining distinct roles: EHD1 in vesiculation vs. EHD3 in tubule stabilization.\",\n      \"method\": \"siRNA knockdown, phospholipase D inhibitor washout assay, site-directed EH domain mutagenesis, co-localization imaging\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — synchronized TRE biogenesis system plus EH domain mutagenesis identifying specific residues, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"27189942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EHD3 binds phosphatidic acid (PA) through its helical domain, as shown by in vitro liposome co-sedimentation. PA–EHD3 interaction induces liposomal tubulation in vitro. Inhibiting PA synthesis with diacylglycerol kinase inhibitor or lysophosphatidic acid acyltransferase inhibitor reduces EHD3-containing tubules and impairs early endosomal trafficking, establishing that PA cooperates with EHD3 to drive membrane tubulation.\",\n      \"method\": \"In vitro liposome co-sedimentation assay, pharmacological PA synthesis inhibition, immunofluorescence tubule counting\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of PA-induced tubulation plus pharmacological loss-of-function in cells, single lab with orthogonal methods\",\n      \"pmids\": [\"26896729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EHD3 accelerates EGFR degradation upon EGF stimulation by increasing EGFR ubiquitination and diverting EGFR trafficking from the recycling route to the degradative pathway. EHD3 reduces endosome-based MAPK and AKT signaling downstream of EGFR without affecting total pathway activity, demonstrating spatial regulation of EGFR signaling.\",\n      \"method\": \"Doxycycline-inducible EHD3 expression, EGFR ubiquitination assay, endosomal trafficking/recycling assays, immunofluorescence, western blot for pathway activation\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple trafficking and signaling assays in a single lab, but no in vitro reconstitution of ubiquitination mechanism\",\n      \"pmids\": [\"27811356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NR5A1 transcriptionally activates EHD3 by binding the conserved 'AGGTCA' sequence in the EHD3 promoter. EHD3 overexpression increases testosterone concentration; EHD3 knockdown decreases testosterone synthesis by reducing endocytosis in Leydig cells. Leydig-cell-specific NR5A1 knockout mice show reduced EHD3, clathrin, and serum testosterone levels.\",\n      \"method\": \"ChIP, dual luciferase reporter assay, siRNA knockdown, exosome tracing/endocytosis assay, conditional NR5A1 KO mouse (CRISPR/Cas9), ELISA\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-validated transcription factor binding plus in vivo conditional KO, single lab\",\n      \"pmids\": [\"33964295\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EHD3 is an EH domain-containing ATPase that localizes to early endosomes and tubular recycling endosomes (TRE), where it functions downstream of a Rab11–Rab8 cascade to mediate early endosome-to-ERC transport, endosome-to-Golgi trafficking, and ciliogenesis initiation via membrane tubulation; it binds phosphatidic acid through its helical domain to drive tubule formation, is SUMOylated on K315/K511 to maintain TRE localization and stability, heterodimerizes with EHD1 through specific EH domain residues (N519/E520) that distinguish its tubule-stabilizing role from EHD1's vesiculation activity, interacts with Rab11-FIP2 and ankyrin-B to traffic cargo including NCX1, Cav1.2, and αvβ3-integrin, and diverts EGFR toward degradative trafficking to attenuate endosomal signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EHD3 is an EH domain-containing membrane-remodeling protein that drives the formation and stabilization of tubular recycling endosomes and governs cargo transit through the early-endosomal system [#0, #11]. It binds phosphatidic acid through its helical domain, and this lipid interaction is sufficient to deform liposomes into tubules in vitro and is required for EHD3-positive tubules and early endosomal trafficking in cells [#12]. Functionally, EHD3 mediates early endosome-to-endocytic recycling compartment (ERC) transport via an EH domain–NPF interaction with the Rab11 effector Rab11-FIP2, a role distinct from EHD1's function in ERC exit [#1]; it also directs early endosome-to-Golgi transport required for Golgi morphology and lysosomal biosynthetic trafficking [#3]. EHD3 heterodimerizes with EHD1 [#0], and the EH domain residues Asn-519/Glu-520 specify its preference for NPF partners and explain its tubule-stabilizing role versus EHD1-driven vesiculation [#11]; SUMOylation at K315 and K511 maintains its localization to tubular ERC structures and recycling activity [#9]. Through these trafficking activities EHD3 controls surface delivery and recycling of specific cargo, including β3/αvβ3-integrin [#6] and the cardiac membrane proteins NCX1 and Cav1.2 in an ankyrin-B–associated complex [#7], and it diverts activated EGFR toward degradation to spatially attenuate endosomal MAPK/AKT signaling [#13]. EHD3, together with EHD1 and the Rab11–Rab8 cascade, also generates ciliary vesicles from distal appendage vesicles at the mother centriole, a tubulation step required for ciliogenesis initiation [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established EHD3 as a membrane-tubulating protein of the recycling system and identified its physical partnership with EHD1, defining a molecular foundation for its trafficking role.\",\n      \"evidence\": \"GFP-fusion localization with domain swapping, yeast two-hybrid and Co-IP in cells\",\n      \"pmids\": [\"12121420\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define the cargo handled by EHD3 tubules\", \"Functional consequence of the EHD1–EHD3 interaction unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed EHD3 binds the Rab11 effector Rab11-FIP2 via EH–NPF contacts and is required for early endosome-to-ERC delivery, distinguishing its trafficking step from EHD1.\",\n      \"evidence\": \"Co-IP with nucleotide-binding mutants, siRNA knockdown, transferrin recycling assay\",\n      \"pmids\": [\"16251358\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of EH–NPF selectivity not resolved\", \"How nucleotide state controls FIP2 binding mechanistically unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Placed EHD3 in a Rab8a/Myosin Vb tubular network distinct from Rab11a compartments, refining the membrane subdomain it occupies.\",\n      \"evidence\": \"FRET and live-cell imaging with co-localization\",\n      \"pmids\": [\"17507647\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"EHD3 role is inferred from co-localization, not direct interaction\", \"Functional contribution to this network untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended EHD3 function beyond recycling to early endosome-to-Golgi transport, linking it to Golgi morphology and lysosomal enzyme delivery.\",\n      \"evidence\": \"siRNA knockdown with Shiga toxin, M6PR, and cathepsin D trafficking assays\",\n      \"pmids\": [\"19139087\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct partner mediating Golgi-directed step beyond rabenosyn-5 not fully defined\", \"Whether tubulation per se drives this route untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected EHD3 expression to cardiac stress, showing it is upregulated across heart failure models downstream of ROS and angiotensin II in parallel with NCX1.\",\n      \"evidence\": \"Western blot across multiple HF models with pharmacological ROS/Ang II manipulation\",\n      \"pmids\": [\"22406195\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ang II/ROS link is pharmacological without genetic dissection\", \"Transcriptional mechanism of upregulation unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated in vivo physiological importance of EHD3 (with EHD4) in glomerular endothelial homeostasis via VEGFR2 recycling.\",\n      \"evidence\": \"Ehd3−/− and Ehd3−/−;Ehd4−/− mouse models with histopathology and receptor localization\",\n      \"pmids\": [\"21408024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct EHD3–VEGFR2 trafficking link is partly inferential\", \"EHD3-specific vs EHD4-redundant contributions not separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified EHD3 as a regulator of rapid β3-integrin recycling and αvβ3-mediated adhesion, defining a specific cargo of its short-loop recycling activity.\",\n      \"evidence\": \"siRNA knockdown, TIRF colocalization, integrin recycling and adhesion assays\",\n      \"pmids\": [\"23781025\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Adaptor coupling EHD3 to integrin not identified\", \"Single lab, single cell type\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a tumor-suppressive function for EHD3 silenced by promoter hypermethylation in glioma, broadening its role to growth and survival control.\",\n      \"evidence\": \"siRNA, inducible re-expression, bisulfite sequencing, 5-Aza demethylation, xenografts\",\n      \"pmids\": [\"24306026\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking trafficking to growth suppression not dissected\", \"Relationship to EGFR/signaling control unexplored here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established EHD3 as essential for cardiac excitability through ankyrin-B–associated trafficking of NCX1 and Cav1.2 to the membrane.\",\n      \"evidence\": \"Cardiac-specific KO mouse, electrophysiology, Ca2+ imaging, Co-IP, localization\",\n      \"pmids\": [\"24759929\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of EHD3/ankyrin-B/cargo assembly unresolved\", \"Whether tubulation activity is required for cardiac cargo delivery untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed SUMOylation at K315/K511 is a post-translational switch controlling EHD3 ERC tubule localization and recycling capacity.\",\n      \"evidence\": \"In vitro SUMOylation, acceptor-lysine mutagenesis, transferrin recycling assays\",\n      \"pmids\": [\"26226295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO E3 ligase and signal triggering SUMOylation unknown\", \"How SUMO controls tubule targeting mechanistically unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined EHD3's role in ciliogenesis initiation, generating ciliary vesicles from distal appendage vesicles via EHD-dependent tubulation within the Rab11–Rab8 cascade.\",\n      \"evidence\": \"siRNA, super-resolution and electron microscopy, live imaging, Co-IP, rescue\",\n      \"pmids\": [\"25686250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"EHD3-specific vs EHD1-redundant contribution to DAV fusion not separated\", \"Direct membrane-fusion machinery coupling not fully mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mechanistically distinguished EHD3 from EHD1 by showing it stabilizes pre-formed tubular recycling endosomes, with EH residues N519/E520 specifying NPF-partner selectivity.\",\n      \"evidence\": \"Synchronized TRE regeneration (PLD inhibitor washout), EH domain mutagenesis\",\n      \"pmids\": [\"27189942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of N519/E520-dependent partner discrimination absent\", \"TRE-stabilizing partners not enumerated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided the biochemical basis for EHD3 tubulation by showing its helical domain binds phosphatidic acid to deform membranes.\",\n      \"evidence\": \"Liposome co-sedimentation, PA synthesis inhibition, tubule quantification\",\n      \"pmids\": [\"26896729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the PA-bound tubulating state lacking\", \"Whether ATPase cycle couples to PA-driven tubulation untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed EHD3 spatially regulates EGFR signaling by promoting receptor ubiquitination and degradative routing, attenuating endosomal MAPK/AKT output.\",\n      \"evidence\": \"Inducible expression, EGFR ubiquitination and trafficking assays, pathway westerns\",\n      \"pmids\": [\"27811356\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of the ubiquitination step\", \"Ubiquitin ligase recruited by EHD3 not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified transcriptional control of EHD3 by NR5A1 and a physiological output in Leydig-cell testosterone synthesis via endocytosis.\",\n      \"evidence\": \"ChIP, luciferase reporter, siRNA, endocytosis assay, conditional NR5A1 KO mouse\",\n      \"pmids\": [\"33964295\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific endocytic cargo driving steroidogenesis not defined\", \"Direct vs indirect contribution of EHD3 to testosterone synthesis unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EHD3's ATPase cycle, PA binding, SUMOylation, and EH–NPF partner selection are integrated into a single tubule-stabilization mechanism, and how this distinguishes the diverse cargo-specific roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural/biochemical model of the tubulation cycle\", \"Determinants of cargo selectivity across cell types unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 11, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0, 1, 3, 6]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 3, 6, 7]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EHD1\", \"RAB11FIP2\", \"ANK2\", \"NCX1\", \"RAB8A\", \"MYO5B\", \"SNAP29\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}